Molecular mechanisms of Mycobacterium tuberculosis resistance to drugs and copper der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades vorgelegt von Alexander Speer aus Witten (geb. in Osnabrück) i Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 12. Juli 2013 Vorsitzender des Promotionsorgans: Prof. Dr. Johannes Barth Gutachter: Prof. Dr. A. Burkovski Prof. Dr. M. Niederweis ii Danke / Thanks Bedanken möchte ich mich zu allererst bei Prof. Dr. Michael Niederweis für die intensive Betreuung dieser Arbeit und seinen wissenschaftlichen Rat. Seine Aufgeschlossenheit und Enthusiasmus zu neuen Projekten und Ideen sorgten immer fuer eine spannende und abwechslungsreiche Arbeit. Ich möchte mich auch bei Prof. Dr. Andreas Burkovski bedanken dessen Hilfsbereitschaft meine Zusammenarbeit mit Dr. Niederweis und die Erstellung dieser Arbeit ermöglichte. Zusätzlich danke ich Ihm und seinem Labor für aussergewöhnlich erfolgreiche Zusammenarbeit an einigen Teilprojekten dieser Studie. Des Weiteren bedanke ich mich bei den Professoren Dr. A. Burkovski, Dr. G. Kreimer, Dr. M. Niederweis und Dr. T. Winkler entsprechend für die Erstellung der Gutachten und die Übernahme der Prüfungspflichten. I would also like to thank all members of the Wolschendorf Lab for their excellent and fruitful collaboration during this work. I am grateful to Dr. Frank Wolschendorf for having always good advice and for his confidence in my work. I would like to thank all current and former members of the Mycolab for creating a wonderful working environment. Special thanks go to Axel Siroy for sharing generously his endless knowledge about proteins with me. I want to thank Jason Huff for his dedicated BSL3 training and expert advice in genomic integration. For reading and adapting countless abstracts I would like to thank Jennifer Rowland. Jim Sun, I would like to thank for introducing me to the work with eukaryotic cell cultures. I am thanking Mikhail Pavlenok for his hospitality during my first year in the US and his technical help during emergencies inside and outside the lab. I would like to thank Olga Danilchanka for her constructive and honest criticism which improved and motivated my work. In addition I am thanking Olga for countless scientific discussions and advice that accelerated my work. I thank Ryan Wells, Virginia Meikle and Doreen William for their willingness to help at all times. Importantly, I thank Ying Wang for keeping the lab stocked. I would like to thank the laboratory of Dr. Sue Michalek for providing us peritoneal macrophages. I am greatful to the laboratory of Dr. Stefan Bossmann for synthesizing the compounds ATSM and GTSM in huge amounts. Von ganzem Herzen bedanke ich mich bei meiner Mutter Elisabeth Speer, die mich all die Jahre so großzügig unterstützt hat und bei meiner Schwester Friederike Speer auf die ich mich immer verlassen konnte. iii Table of Contents 1 Zusammenfassung.................................................................................................................. 1 1 Summary ................................................................................................................................ 3 2 Introduction ............................................................................................................................. 5 2.1 Clinical relevance of M. tuberculosis ................................................................................. 5 2.2 The mycobacterial cell envelope ....................................................................................... 6 2.2.1 Structure of the cell envelope of mycobacteria ........................................................... 6 2.2.2 Cell wall biosynthesis ................................................................................................. 8 2.2.3 Mycobacterial lipids and lipoglycans ........................................................................... 9 2.2.4 Proteins of the mycobacterial outer membrane .......................................................... 9 2.3 Pathogenesis ...................................................................................................................10 2.3.1 Innate immune system during bacterial infection .......................................................10 2.3.2 M. tuberculosis adaptation to an intracellular lifestyle ................................................11 2.4 Dual role of copper in bacteria .........................................................................................12 2.5 Role of copper during M. tuberculosis infection................................................................12 2.6 Tuberculosis drug development .......................................................................................15 2.7 Goal of this thesis ............................................................................................................16 3 Results ...................................................................................................................................17 3.1 Characterization of the rv1697-rv1698 operon in M. tuberculosis ....................................17 3.1.1 Rv1697 and rv1698 are encoded in one operon ........................................................17 3.1.2 Subcellular localization of Rv1697 and Rv1698 .........................................................18 3.1.2.1 Ms3748 and Ms3747 are membrane associated proteins ...................................18 3.1.2.2 Ms3748 and Ms3747 interact in vivo ...................................................................19 3.1.2.3 Ms3748 is translocated into the periplasm ..........................................................20 3.1.3 Characterization of rv1697 and rv1698 mutants and their homologs .........................22 3.1.3.1 Construction of rv1697Δcth, ms3747Δcth and ΔcgR_1476 mutant strains ..........22 3.1.3.2 Growth and morphological characterization of rv1697Δcth and ms3747Δcth ......25 3.1.3.3 Susceptibility of the ms3748Δcth mutant strains to SDS, copper and Malachite green ..............................................................................................................................27 3.1.3.4 Accumulation of Congo red, chenodeoxycholate and ethidium bromide .............29 3.1.3.5 Antibiotic sensitivity of rv1697Δcth and ms3748Δcth strains ...............................30 3.1.4 Cell wall analysis of ms3748Δcth and ΔcgR_1476 deletion strains ...........................32 3.1.4.1 Whole cell lipid analysis ......................................................................................32 iv 3.1.4.2 Membrane vesicles from culture filtrate ...............................................................33 3.1.4.3 Differences in cell wall sugar content ..................................................................35 3.2 Screening for copper sensitivity enhancing compounds ...................................................36 3.2.1 Assay development and optimization ........................................................................36 3.2.1.1 Media optimization ..............................................................................................36 3.2.1.2 Determination of the optimal copper concentration .............................................37 3.2.2 Assay validation and establishment of key assay parameters ...................................38 3.2.2.1 Effect of neocuproine on M. tuberculosis ............................................................38 3.2.2.2. Determination of Z’-factors .................................................................................39 3.2.3 Limited pilot screen and analysis of hits ....................................................................40 3.2.3.1 Synergy between copper ions and Bis-dithiosemicarbazones .............................40 3.2.3.2 Activity of diacetylbis(N(4)-methyl-3-thiosemicarbazone and glyoxalbis(N(4)- methyl-3-thiosemicarbazone against non-growing M. tuberculosis .................................40 3.2.3.3 Therapeutic index of diacetylbis(N(4)-methyl-3-thiosemicarbazone and glyoxalbis(N(4)-methyl-3-thiosemicarbazone ..................................................................42 4 Discussion .............................................................................................................................44 4.1 Rv1697 is an essential core protein of M. tuberculosis required for cell wall integrity .......44 4.1.1 Rv1697 of M. tuberculosis and its homologs in M. smegmatis and C. glutamicum have the same function ...............................................................................................................44 4.1.2 Ms3748 and Ms3747 contribute to cell wall integrity ..................................................44 4.1.3 Effects of ms3748Δcth mutations on the mycobacterial cell wall ...............................45 4.1.4 The functions of Rv1697 and Rv1698 are connected ................................................46 4.1.5 Rv1697 is a potential drug target for TB chemotherapy .............................................47 4.1.6 Putative alternative function of Rv1697 .....................................................................48 4.2 Copper-boosting compounds: a novel concept for anti-mycobacterial drug discovery .....49 4.2.1 Assay development for new anti-TB drugs ................................................................49